A central paradigm for eukaryotic gene control posits that sequence-specific transcription factors search for and locate their genomic targets in a crowded nuclear environment, often acting combinatorially and cooperatively at regulatory DNA elements in chromatin to recruit and direct the ordered assembly of a transcription pre-initiation complex composed of general transcription factors and RNA polymerase. Although the identities and functions of several hundred transcription-related proteins are known, there is little information on the timescales under which they operate in the transcription process, and the regulatory influence of chromatin architecture, remodeling and modification on transcription protein kinetics. This proposal aims to test the hypothesis that chromatin binding of transcription initiation proteins in the cell nucleus occurs with rapid kinetics in live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics. We will use live-cell single-molecule imaging in the model organism budding yeast to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution. This ‘in vivo biochemistry’ approach differs from and is complementary to ChIP-Seq techniques that map transcription factor occupancy genome-wide at base pair resolution but provide little information on binding dynamics. To elucidate regulatory contributions of chromatin architecture to transcription initiation protein dynamics, we will measure their mobilities in yeast mutants conditionally depleted for chromatin remodeling and modification enzymes. We will engineer and functionally validate DNA constructs encoding components representative of the general transcription factors and major sequence-specific DNA binding transcription factors fused to a self- labeling protein tag (HaloTag) that allows labeling with a cell-permeable organic fluorophore (Janelia Fluor). Live- cell imaging of fluorescently labeled transcription factors at single-molecule resolution will measure protein diffusion and distinguish between chromatin-bound and chromatin-free populations and estimate residence times of the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the diffusive parameters of transcription initiation proteins under conditions of chromatin perturbation to inform which among several diffusive parameters are subject to chromatin controls. By combining with conditional mutant genetics with live-cell single-molecule imaging, we hope to transform understanding of the kinetic mechanisms by which chromatin architecture regulates the transcription initiation process and develop a potentially routine technology complementary to current genome-wide analytical techniques to benefit other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.